Plants adapt to various types of environmental stress such as the temperature and salt of their habitats. However, in terms of temperature stress, for example, plants are susceptible to hot or cold temperatures when exposed to environments over or under the maximum or minimum optimum growth temperature, leading to impairment upon the gradual or sudden loss of the physiological functions of cells. Efforts have been made to expand the temperature adaptability of plants by breeding means such as selection or cross breeding in order to make use of wild plants adapted to various temperature environments for food crops, horticultural plants, and the like. The planting period in which vegetables, flowers and ornamental plants, fruit trees, and the like can be cultivated has been expanded by such breeding means as well as by protected horticulture. However, Japan in particular extends a considerable length to the north and south, with extreme variation in climate and considerable change in seasons from area to area, resulting in a greater risk of crop exposure to temperature environments that are not conducive to growth, depending on the area and season. Rice, for example, which originates in tropical regions, can now be cultivated in cooler areas such as the Tohoku district and Hokkaido as a result of improvements in varieties since the Meiji period, and are now cultivated as staples of these regions, but unseasonably low temperatures in early summer in these areas recently have resulted in cold-weather damage, leading to the problem of severe shortages even now. Recently, abnormal atmospheric phenomena attributed to global warming or El Nino have resulted in major crop damage, and the rice shortages caused by severe cold-weather damage in 1993 are still remembered. Culinary plants include many crops of tropical origin among fruits and vegetables such as tomatoes, cucumbers, melons, and water melon. Such crops are in high demand and are extremely important in terms of agriculture, and they have long been involved in greenhouse culture. However, since the oil shock of 1974, the conservation of resources and lowering warming costs have become a problem. The conservation of resources in protected horticulture has been studied from a variety of perspectives, from the structural concerns of green houses to cultivation techniques, but the most basic consideration is increasing the cold tolerance of crops.
In regard to salt stress, it is said that about 10% of all land surface area is salt damaged, and the spread of saline soil, primarily in arid areas such as Southeast Asia and Africa is becoming a serious agricultural problem.
Water stress can be a major form of stress for plants, and is significantly affected by the amount and distribution of precipitation when temperature is not a limiting factor. The growth and yield of crops is tremendously dependent upon drought stress in semi-arid regions and the like which are important areas of crop cultivation.
Cross breeding, breeding making use of recent genetic engineering techniques, methods making use of the action of plant hormones and plant regulators, and the like have been employed to improve tolerance against these various types of environmental stress.
Environmental stress-tolerant plants have thus far been produced using genetic engineering techniques. Genes reported to have been used in the improvement of cold tolerance include fatty acid desaturase genes of membrane lipids (ω-3 desaturase gene, glycerol-3-phosphate acyltransferase gene, and stearoyl-ACP-desaturase gene), pyruvic-phosphate dikinase genes involved in photosynthesis, and genes coding for proteins with cryoprotection/prevention activity (COR15, COR85, and kin1).
Genes reported to have been used in the improvement of tolerance against salt stress and water stress include glycine betaine synthetase genes of osmotic regulators (choline monooxygenase gene and betaine aldehyde hydrogenase gene) and proline synthetase genes (1-pyroline-5-carboxylate synthetase).
Genes reported to have been used in the improvement of tolerance against herbicidal stress include aromatic amino acid synthetase genes (5-enol-pyruvylshikimate-3-phosphate synthase gene) and detoxification enzyme genes (nitrilase gene and phosphinothricin acetyltransferase gene). Although plants involving transformants of such genes have been of some practical use in herbicidal stress-tolerant plants, most have not been effective enough to be used for actual industrial purposes and have not been put to practical use.
Polyamines, the general term for aliphatic hydrocarbons with 2 or more primary amino groups, are ubiquitous natural substances in organisms, with more than 20 types discovered so far. Typical polyamines include putrescine, spermidine, and spermine. The known primary physiological action of polyamines includes (1) nucleic acid stabilization and structural modification through interaction with nucleic acids; (2) promotion of various nucleic acid synthesis systems; (3) activation of protein synthesis systems; and (4) stabilization of cell membranes and enhancement of membrane permeability of substances. Reports on the role of polyamines in plants include cell protection and promotion of nucleic acid or protein biosynthesis during cellular growth or division.
The involvement of polyamines in various types of environmental stress has recently been reported. They have been implicated in cold stress (J. Japan Soc. Hortic. Sci., 68, 780-787 (1999); J. Japan Soc. Hortic. Sci., 68, 967-973 (1999); Plant Physiol. 124, 431-439 (2000)); salt stress (Plant Physiol. 91, 500-504 (1984)); acid stress (Plant Cell Physiol. 38(10), 1156-1166 (1997)); osmotic stress (Plant Physiol. 75, 102-109 (1984)); pathogen infection stress (New Phytol., 135, 467-473 (1997)); and herbicidal stress (Plant Cell Physiol. 39(9), 987-992 (1998)), but all of these reports assume the involvement of polyamines based on the correlation between growth reaction or stress tolerance and changes in polyamine concentration, yet report nothing on their involvement at the genetic level between environmental stress and polyamine metabolism-related enzyme genes coding for polyamine metabolism-related enzymes.
Known polyamine metabolism-related enzymes involved in the biosynthesis of plant polyamines include arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), spermidine synthase (SPDS), and spermine synthase (SPMS). Several polyamine metabolism-related genes coding for such polyamine metabolism-related enzymes have already been isolated from plants. The ADC gene has been isolated from oats (Mol. Gen., Genet. 224, 431-436 (1990)), tomatoes (Plant Physiol., 103, 829-834 (1993)), Arabidopsis thaliana (Plant Physiol., 111, 1077-1083 (1996)), and peas (Plant Mol. Biol., 28, 997-1009 (1995)); the ODC gene has been isolated from datura (Biochem. J., 314, 241-248 (1996)); the SAMDC gene has been isolated from potatoes (Plant Mol. Biol., 26, 327-338 (1994)), spinach (Plant Physiol., 107, 1461-1462 (1995)), and tobacco; and the SPDS gene has been isolated from Arabidopsis thaliana (Plant Cell Physiol., 39(1), 73-79 (1998)).
An object of the present invention is thus to undertake biochemical analysis in cultivars having high and low environmental stress tolerance so as to elucidate the mechanism intimately involved in environmental stress tolerance. An object is thus to produce recombinant plants with improved environmental stress tolerance by screening genes that play a major role in this mechanism and by artificially controlling the expression of such genes to lower polyamine levels.
More specifically, the mechanism intimately involved in chilling tolerance is elucidated through biochemical analysis of cultivars having chilling-tolerant and chilling sensitive. Genes playing a major role in this mechanism are obtained. The genes are then applied to actual plants to check the effects at the practical level. Although polyamine metabolism-related genes have been isolated from various plants thus far, there has been little research on the relation between polyamine metabolism-related genes and cold tolerance, and no polyamine metabolism-related genes whose level of expression changes when exposed to low temperature have been found. An object of the invention is thus to provide polyamine metabolism-related genes deeply involved in cold tolerance, and to use such genes to produce plants which are more tolerant to various types of environmental stress such as cold tolerance.